Long non-coding RNAs (lncRNAs) are ubiquitously expressed RNA molecules of more than 200 nucleotides without substantial ORFs. LncRNAs could act as epigenetic regulators of gene expression affecting transcription, mRNA stability and transport, and translation, although, precise functions have been attributed to only few of them. Competing endogenous RNAs (ceRNAs) represent one recently emerged type of functional lncRNAs that share microRNA recognition sequences with mRNAs and may compete for microRNA binding and thus affect regulation and function of target mRNAs. We studied the epigenetic regulation of the BARD1 gene. The BARD1 protein acts as tumor suppressor with BRCA1. In cancer, mRNAs encoding the tumor suppressor full length BARD1 are often down-regulated while the expression of oncogenic truncated isoforms is boosted. We found that the BARD1 3′UTR is almost 3000 nt long and harbors a large number of microRNA binding elements. In addition we discovered a novel lncRNA, BARD1 9′L, which is transcribed from an alternative promoter in intron 9 of the BARD1 gene and shares part of the 3′UTR with the protein coding BARD1 mRNAs. We demonstrate with the example of two microRNAs, miR-203 and miR-101, that they down-regulate the expression of FL BARD1 and cancer-associated BARD1 mRNAs, and that BARD1 9′L counteracts the effect of miR-203 and miR-101, As BARD1 9′L is abnormally over-expressed in human cancers, we suggest it might be a tumor promoting factor and treatment target.
Long non coding RNAs (lncRNA) are RNA molecules longer than 200 nucleotides and without substantial ORFs that may encode polypeptides (Kapranov et al. 2007; Dinger et al. 2009). The GENCODE consortium working within the framework of the ENCODE project recently presented the annotation of human lncRNAs, including 9277 genes which produce 14,880 transcripts (Derrien et al. 2012). Interestingly, they demonstrated that lncRNAs coding genes have histone-modification profiles, splicing signals, and exon/intron lengths similar to that of protein-coding genes. LncRNAs may also be polyadenylated and share exon sequences with the protein coding genes (Carninci et al. 2005; Derrien et al. 2012). LncRNAs are found in the nucleus as well as in the cytosol, and many lncRNAs are tissue specifically expressed (Carninci et al. 2005; Birney et al. 2007; Derrien et al. 2012). Despite the ubiquitous presence and abundant expression of lncRNAs, a function was attributed to only few of them.
LncRNAs are involved in the epigenetic regulation of gene expression, as was shown with the examples of the regulation of the HOXC gene by antisense HOX intergenic RNA (HOTAIR) (He et al. 2011) and X chromosome inactivation in female mammals mediated by the inactive X-specific transcript (XIST) (Brown et al. 1991). LncRNAs may also play a role in a variety of cellular processes including transcription regulation, alternative splicing, RNA decay, nuclear import, and translation (Ponting et al. 2009; Wilusz et al. 2009; Wapinski and Chang 2011).
Competing endogenous RNAs (ceRNA) represent one of the recently emerged types of functional lncRNAs. It has been shown that lncRNAs that share microRNA recognition elements (MRE) with specific mRNAs may compete for microRNA binding and thus affect the function of these mRNAs. The striking example of such a competing endogenous RNA (ceRNA) is a ˜500 nt lncRNA first identified as the most up-regulated gene in hepatocellular carcinoma and colorectal cancers (Panzitt et al. 2007; Matouk et al. 2009). This RNA, termed HULC (highly up-regulated in liver cancer), is polyadenylated and consists of two exons. It inhibits the activity and competes for binding of miR-372 and reduces the activity of its target gene PRKACB (Wang et al. 2010). Similar to HULC, the non-coding PTENP1 pseudogene RNA, regulates tumor suppressor gene PTEN acting as ceRNA (Poliseno et al. 2010). PTENP1 mRNA shares homology with the PTEN mRNA 3′UTR and competes for microRNAs that down-regulates PTEN expression. Knockdown of endogenous PTENP1 in prostate cancer cells results in an increase in PTEN mRNA and protein levels and those of the miR-17-5 p/20 target p21 and potentially other relevant targets. A similar correlation of expression is found between KRAS and its pseudogene KRAS1P (Poliseno et al. 2010). It was suggested that protein-coding mRNAs and lncRNAs can interact with each other competing for microRNA binding (Salmena et al. 2011). CeRNAs are thus lncRNAs that are particularly interesting considering the importance of the regulatory function of microRNAs.
Indeed, microRNAs, small evolutionarily conserved RNAs of 18-25 nucleotides, act as expression regulators of genes involved in fundamental processes, such as development, differentiation, proliferation, survival and death (Ambros 2004). Researchers in the field estimate that there are likely more than a thousand microRNAs in the human genome, and that these microRNAs may target up to one-third of all human genes (Croce 2009). A mature microRNA is loaded into the microRNA-induced silencing complex where it is believed to either repress mRNA translation or reduce mRNA stability following imperfect binding between the microRNA and MRE, typically within the 3′ UTR of target genes (Garzon et al. 2010). MicroRNAs may function as tumor suppressors, oncogenes, or both. In many cases, these functions are disease or tissue-specific. Several observations implicated global deregulation of microRNAs in both solid and hematological malignancies (Croce 2009; Nana-Sinkam and Croce 2011).
In this study we show that the BRCA1-associated RING domain protein 1 (BARD1) gene expression may be regulated by a large number of microRNAs and by a presumed lncRNA competing for microRNA binding. BARD1 has tumor suppressor functions and is involved in a number of cellular processes including DNA repair, transcriptional regulation, chromatin remodeling, cell cycle checkpoint control, and mitosis (Jin et al. 1997; Hashizume et al. 2001; Westermark et al. 2003; Starita and Parvin 2003; Irminger-Finger and Jefford 2006; Joukov et al. 2006; Laufer et al. 2007; Murray et al. 2007; Ryser et al. 2009; Larsen et al. 2010; Li and Yu 2013). BARD1 has also been shown to be essential for the maintenance of genomic stability (Irminger-Finger et al. 1998; McCarthy et al. 2003; Li and Yu 2013). Several protein-coding mRNA isoforms of variable exon composition are expressed in human and murine cancers (Feki et al. 2005; Wu et al. 2006a; Li et al. 2007b; Lombardi et al. 2007; Sporn et al. 2011; Zhang et al. 2012a, 2012b). The full length (FL) BARD1 mRNA includes 11 exons (FIG. 1A) and encodes a protein comprising an N-terminal RING-finger domain, three ankyrin repeats (ANK), and two C-terminal BRCT domains. In cancer, the mRNA encoding tumor suppressor FL BARD1 is often down-regulated, while the expression of other splice isoforms is boosted. The overexpression of BARD1 isoforms that lack RING or RING and ANK was not only associated with breast, ovarian, endometrial, cervical, lung, and colon cancer (Wu et al. 2006a; Li et al. 2007b; Sporn et al. 2011; Zhang et al. 2012a, 2012b), but also correlated with advanced cancer stages of breast and ovarian cancer (Li et al. 2007b) and decreased patient survival time in lung cancer (Zhang et al. 2012a). Many studies suggest that the deficiency of FL BARD1 may have an oncogenic effect (Irminger-Finger et al. 1998; McCarthy et al. 2003; Tsuzuki et al. 2006; Capasso et al. 2009; Sabatier et al. 2010; Sporn et al. 2011) which would be consistent with the expression of spliced isoforms (Li et al. 2007a; Ryser et al. 2009; Ratajska et al. 2011; Bosse et al. 2012).
Importantly, SNPs in non-coding regions of or close to the BARD1 gene were clearly associated with neuroblastoma (Capasso et al. 2009; Nguyen et al. 2011; Latorre et al. 2012; Lee et al. 2013), and expression of isoforms was upregulated in the neuroblastoma-associated SNP genotype and correlated with disease progression and poor outcome (Bosse et al., 2012). In this study we provide evidence that a lncRNA expressed from an alternative intronic promoter of BARD1 may positively regulate BARD1 isoform expression.